Duong, Chau Minh

English

Clark University

Clark University Clark Digital Commons Undergraduate Student Academic Festivals ClarkFEST Fall 2022 Oct 1st, 12:00 AM The Effects of tRNA Methylation on Protein Synthesis in Dictyostelium discoideum Chau Minh Duong Clark University, cduong@clarku.edu Follow this and additional works at: https://commons.clarku.edu/asdff (2022). The Effects of tRNA Methylation on Protein Synthesis in Dictyostelium discoideum. Retrieved from https://commons.clarku.edu/asdff/clarkfestfall2022/Posters/32 This Open Access Event is brought to you for free and open access by the Student Works at Clark Digital Commons. It has been accepted for inclusion in Undergraduate Student Academic Festivals by an authorized administrator of Clark Digital Commons. For more information, please contact larobinson@clarku.edu, cstebbins@clarku.edu. a)c)Figure 8: a) Primary screening of entry clones (D-TOPO vector + codon repeats) using restrictiondigest with EcoRV-a single-cutter enzyme. b,c)Secondary screening of entry clones using PCR.Positive control is indicated as +, and negativecontrol is indicated as -. Clear bands within theexpected size were marked with red box.b)The Effects of tRNA Methylation on Protein Synthesis in Dictyostelium discoideumChau Duong ‘22, Professor Denis A. LarochelleBiology Department - Summer Undergraduate Research Experience (SURE) Award RecipientIntroductionEpitranscriptomics describes RNA modifications leading to changes in gene expression. This is apotential research avenue in studying therapeutic targets for cancer. In this research project, we focuson studying how methylating transfer RNA (tRNA - which delivers amino acids during translation)will affect protein synthesis. Dnmt2/Trdmt1 (tRNA aspartic acid methyltransferase) in humans and DnmA in Dictyosteliumdiscoideum (D.discoideum) are highly conserved, homologous enzymes that perform tRNAmethylation (1,2). Structural analysis of human Dnmt2 reveals a binding site for SAM- a cofactorin the methyltransferase reaction. DnmA mutant cells (DnmA KO) showed abnormal cell morphologies: multiple sori, larger cellsize, multiple centrosomes (3). Gene ontology revealed that this enzyme is also linked with othercomponents regulating various cellular processes. Codon usage bias is the preference among synonymous codons in protein synthesis. This is ahighly conserved phenomenon across eukaryotes. Codon usage bias can also affect geneexpression by controlling translation efficiency.Research Questions Does tRNA methylation by DnmA affect protein synthesis? If so, what arethe molecular targets?What is the relationship between DnmA tRNA methylation activity andcodon usage bias?Methods1. Generate 3 vector DNA constructs, each with a stretch of 8 glycine repeats of one of the 3glycine codons (GGT, GGC, GGA) D-TOPO Reaction: Insert 8 glycine repeats of GGT/GGC/GGA codons into pENTRTM/ D-TOPO vector LR Reaction: Insert D-TOPO vector contained glycine repeats into destination vectors to fusethe repeat with a Green Fluorescence Protein (GFP) tag or a Red Fluorescence Protein(mRFP) tag2. Confirm the vector cloning by restriction digest, PCR, and Sanger gene sequencing Restriction digest with a single-cutter enzyme: EcoRV or ApaI. PCR with M13 primer and the insert primer to amplify the inserted sequence. The expectedband on gel electrophoresis is 156bp. Positive clones expressing the inserted band will be confirmed with gene sequencing.Preliminary Results Successfully generated the designed DNA reporter constructs, each with 8 repeats ofGGT/GGC/GGA and a fluorescent tag (Figure 9,10,11). The PCR results alone could not distinguish the orientation of the insert (Figure 8). Theconstructs were confirmed with Sanger sequencing. The first transfection showed no fluorescent signal in both the control and DnmA - KO cells.Vector Cloning1. Express DNA constructs in wild-type (AX4strain) and DnmA-KO D.discoideum celllines. Normal protein synthesis of theconstructs will express GFP/mRFP. Cellswill be observed under fluorescencemicroscopy and measured for fluorescenceintensity using FACS (Fluorescence-activated cell sorting flow cytometry).2. Using R programming to screen for glycine-rich protein targets from D.discoideumcoding sequence database. If the effects oftRNA methylation are confirmed, we willinvestigate the potential molecular targets,including glycine-rich proteins.Protein Expression & Molecular Target IdentificationFuture DirectionsOptimize the transfection protocols.Observe the transfected cells with fluorescence microscopy and confirm the GFP expressionwith Western Blotting. Track the morphological and phenotypical differences (size, shape, growth rate) between AX4and DnmA-KO cells.Assess the potential protein targets based on their genetic conservation and gene ontology .Acknowledgements:Figure 4: Gene ontology predictions of potential interactors of dnmA and their associated molecular functions. Data were obtained from STRING Database ver. 11.5Figure 5: Vector cloning mapand codon repeat insertdesign. This working schemeincludes:1- inserting the DNAdesigned construct into theD-TOPO vector,2- transferring the insertfrom D-TOPO to thedestination vector containingthe fluorescence tag usingthe LR reaction.(Image was created usingBiorender.com)Figure 7: Example of 10/34 results of glycine-rich proteins containing a stretch of 10X glycine obtainedwith R. The search includes a number order for the sequence, number of 10xG stretch and the sequence/geneID.Figure 1: tRNA in the translation process (Source: NIH)Figure 3: a) 3D Structure of human Dnmt2- (generated using MOE Program 2020, PDB-ID:1G55); b) Reaction mechanism of methylation of cytosine by Dnmt2 with SAM cofactor (generated with ChemDraw Program)a) b)Figure 2: Glycine codon usage frequency inD.discoideum. Data were collected from the CodonUsage Database- Nakamura Y. (2000).Figure 6: Predictions of protein expression in wild-type(AX4) and DnmA KO D.discoideum cells (Image wascreated using Biorender.com)I would like to express my special thanks to Professor Denis A. Larochelle for research guidance and sponsorship in thisresearch project. I also appreciate the help and advice from Professor Javier F. Tabima, and the former and current labmembers: Zaza Gelashvili, Davin Tafuri, and Eun-Mi Jeong,. I want thank the Biology Department - Clark University for theSummer Undergraduate Research Experience (SURE) award. I would also like to acknowledge the Gustaf H. Carlson Schoolof Chemistry and Biochemistry - Clark University for providing access to MOE (Molecular Operating Environment) Program.Figure 9: Gene sequencing chromatogram for GGT-repeat DNA fused with GFP tag.Figure 10: Gene sequencing chromatogram for GGC-repeat DNA fused with GFP tag.Figure 11: Gene sequencing chromatogram for GGA-repeat DNA fused with GFP tag.1. T. P. Jurkowski et al., Human DNMT2 methylates tRNA(Asp) molecules using a DNA methyltransferase-like catalytic mechanism. Rna 14, 1663-1670 (2008).2. S. Müller et al., Target recognition, RNA methylation activity and transcriptional regulation of the Dictyostelium discoideum Dnmt2-homologue (DnmA). Nucleic Acids Res 41, 8615-8627 (2013).3. Zaza Gelashvili, Determining the Effects of Methylation by DnmA in Dictyostelium discoideum. Master of Science Thesis (2020).4. Nakamura, Y., Gojobori, T. and Ikemura, T, Codon usage tabulated from the international DNA sequence databases: status for the year 2000. Nucl. Acids Res. 28, 292. (2000)References:

The Effects of tRNA Methylation on Protein Synthesis in Dictyostelium discoideum

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The Effects of tRNA Methylation on Protein Synthesis in Dictyostelium discoideum

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